Bottom Line:
Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes.These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body.In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes.

ABSTRACTIn mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.

pgen-1003538-g004: Relocalisation of persistent radiation-induced DSBs into a pseudo XY body.(A) Irradiated Spo11YF/YF spermatocytes were collected 1 h, 48 h and 120 h upon irradiation and immunostained for RAD51 (green), SYCP3 (red), and γH2AX (blue). Spermatocytes that were irradiated at the leptotene stage, should have reached zygotene and pachytene with respect to the pattern of γH2AX, at 48 and 120 h following irradiation, respectively. (B) Fraction of cells showing a pseudo XY body upon irradiation at the analysed time-points (n = 50). (C) Immunostaining of Spo11YF/YF spermatocyte 120 hours after irradiation with anti-RNA pol II (green) and anti-γH2AX (red). The intense γH2AX domain (pseudo XY body) corresponds to a nuclear area depleted for RNA pol II.

Mentions:
The localised presence of DNA repair foci in one or a few pseudo XY bodies indicates that DNA damage in spermatocytes tends to concentrate in a single, transcriptionally silenced area. To test this hypothesis, we induced exogenous DSBs in Spo11YF/YF spermatocyte nuclei by whole-body irradiation, and analysed the presence of DSB markers at different time points following the treatment. We observed approximately 120 (±5.3, n = 30) RAD51 foci and a nucleus-wide accumulation of γH2AX at 1 h following irradiation. Interestingly, 48 hours after irradiation, we still observed extensive H2AX phosphorylation emanating from the many RAD51 foci (Figure 4A). However, 120 h following irradiation, when cells that were irradiated at leptotene would have progressed to pachytene in a wild type background, a pseudo XY body was observed in about 90% (n = 70) of the analysed nuclei (Figure 4B). These pseudo XY bodies always contained RAD51 foci (25.1±1.73, n = 50), and the majority of the radiation-induced RAD51 foci that are still present at this time point (65.7%) localized in the pseudo XY body (Figure 4A). These data show that the persistent radiation-induced DSBs tend to relocalize in a specific nuclear subdomain. This phenomenon is in accordance with the colocalization of unsynapsed or partially synapsed translocation chromosomes, carrying persistent meiotic DSBs, with the XY body [38].

pgen-1003538-g004: Relocalisation of persistent radiation-induced DSBs into a pseudo XY body.(A) Irradiated Spo11YF/YF spermatocytes were collected 1 h, 48 h and 120 h upon irradiation and immunostained for RAD51 (green), SYCP3 (red), and γH2AX (blue). Spermatocytes that were irradiated at the leptotene stage, should have reached zygotene and pachytene with respect to the pattern of γH2AX, at 48 and 120 h following irradiation, respectively. (B) Fraction of cells showing a pseudo XY body upon irradiation at the analysed time-points (n = 50). (C) Immunostaining of Spo11YF/YF spermatocyte 120 hours after irradiation with anti-RNA pol II (green) and anti-γH2AX (red). The intense γH2AX domain (pseudo XY body) corresponds to a nuclear area depleted for RNA pol II.

Mentions:
The localised presence of DNA repair foci in one or a few pseudo XY bodies indicates that DNA damage in spermatocytes tends to concentrate in a single, transcriptionally silenced area. To test this hypothesis, we induced exogenous DSBs in Spo11YF/YF spermatocyte nuclei by whole-body irradiation, and analysed the presence of DSB markers at different time points following the treatment. We observed approximately 120 (±5.3, n = 30) RAD51 foci and a nucleus-wide accumulation of γH2AX at 1 h following irradiation. Interestingly, 48 hours after irradiation, we still observed extensive H2AX phosphorylation emanating from the many RAD51 foci (Figure 4A). However, 120 h following irradiation, when cells that were irradiated at leptotene would have progressed to pachytene in a wild type background, a pseudo XY body was observed in about 90% (n = 70) of the analysed nuclei (Figure 4B). These pseudo XY bodies always contained RAD51 foci (25.1±1.73, n = 50), and the majority of the radiation-induced RAD51 foci that are still present at this time point (65.7%) localized in the pseudo XY body (Figure 4A). These data show that the persistent radiation-induced DSBs tend to relocalize in a specific nuclear subdomain. This phenomenon is in accordance with the colocalization of unsynapsed or partially synapsed translocation chromosomes, carrying persistent meiotic DSBs, with the XY body [38].

Bottom Line:
Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes.These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body.In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes.

ABSTRACTIn mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.